The invention relates to the field of microbiology. More specifically, the invention relates to the use of a novel methanotrophic bacterial strain capable of utilizing a central carbon pathway for more efficient production of commercially useful products.
Methanotrophic bacteria are defined by their ability to use methane as their sole source of carbon and energy. Although methanol is an obligate intermediate in the oxidation of methane, the ability to grow on methanol alone is highly variable among the obligate methanotrophs due to its toxicity (Green, Peter. Taxonomy of Methylotrophic Bacteria. In: Methane and Methanol Utilizers (Biotechnology Handbooks 5) J. Colin Murrell and Howard Dalton eds. 1992 Pleanum Press NY, pp. 23-84). Methane monooxygenase is the enzyme required for the primary step in methane activation and the product of this reaction is methanol (Murrell et al., Arch. Microbiol. (2000), 173(5-6), 325-332). This reaction occurs at ambient temperatures and pressures, whereas chemical transformation of methane to methanol requires temperatures of hundreds of degrees and high pressure (Grigoryan, E. A., Kinet. Catal. (1999), 40(3), 350-363; WO 2000007718; U.S. Pat. No. 5,750,821). It is this ability to transform methane under ambient conditions along with the abundance of methane that makes the biotransformation of methane a potentially unique and valuable process.
The commercial applications of biotransformation of methane have historically fallen broadly into three categories: 1) Production of single cell protein, (Sharpe D. H. BioProtein Manufacture (1989). Ellis Horwood series in applied science and industrial technology. New York: Halstead Press) (Villadsen, John, Recent Trends Chem. React. Eng., [Proc. Int. Chem. React. Eng. Conf.], 2nd (1987), Volume 2, 320-33. Editor(s): Kulkarni, B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher: Wiley East., New Delhi, India; Naguib, M., Proc. OAPEC Symp. Petroprotein, [Pap.] (1980), Meeting Date 1979, 253-77 Publisher: Organ. Arab Pet. Exporting Countries, Kuwait, Kuwait); 2) epoxidation of alkenes for production of chemicals (U.S. Pat. No. 4,348,476); and 3) biodegradation of chlorinated pollutants (Tsien et al., Gas, Oil, Coal, Environ. Biotechnol. 2, [Pap. Int. IGT Symp. Gas, Oil, Coal, Environ. Biotechnol.], 2nd (1990), 83-104. Editor(s): Akin, Cavit; Smith, Jared. Publisher: Inst. Gas Technol., Chicago, Ill.; WO 9,633,821; Merkley et al., Biorem. Recalcitrant Org., [Pap. Int. In Situ On-Site Bioreclam. Symp.], 3rd (1995), 165-74. Editor(s): Hinchee, Robert E; Anderson, Daniel B.; Hoeppel, Ronald E. Publisher: Battelle Press, Columbus, Ohio: Meyer et al., Microb. Releases (1993), 2(1), 11-22). Only epoxidation of alkenes has experienced little commercial success due to low product yields, toxicity of products and the large amount of cell mass required to generate products.
Large-scale protein production from methane, termed single cell protein or SCP has been technically feasible and commercialized at large scale (Villadsen supra). However, SCP has been less than economically successful due to the relatively high cost to produce microbial protein compared to agriculturally derived protein (i.e. soy protein). Single cell protein is a relatively low value product and therefore economic production cannot tolerate heavy bioprocessing costs. For this reason the yield of the methanotrophic strain may be critical to the overall economic viability of the process. Microbial biomass produced by methanotrophic bacteria is typically very high in protein content (xcx9c70-80% by weight), which can restrict the direct use of this protein to certain types of animal feed.
The conversion of C1 compounds to complex molecules with Cxe2x80x94C bonds is a difficult and capital intensive process by traditional chemical synthetic routes. Traditionally, methane is first converted to synthesis gas (mixtures of hydrogen, carbon monoxide and carbon dioxide), which is then used to produce other small molecular weight industrial precursors. Typically these are xe2x80x9ccommodityxe2x80x9d type chemicals such as acetate, formaldehyde, or methanol. The basic problem is activation of the methane molecule which is thermodynamically very difficult to achieve by chemical means. xe2x80x9cActivationxe2x80x9d refers to the process of making the chemically unreactive methane molecule more reactive.
Methanotrophic bacteria contain enzymes (methane monooxygenases) which are capable of methane activation at ambient temperatures and pressures. Methane activation consists of oxygen insertion into methane to form methanol which is much more readily metabolized to more complex molecules within the cell. Two types of methane monooxygenase are found in methanotrophic bacteria. A particulate methane monooxygenase (pMMO) has a narrow substrate specificity and is incapable of oxygen insertion into more complex molecules. Some, but not all methanotrophs may also contain a soluble methane monooxygenase (sMMO). This enzyme has been the subject of much investigation and proprietary claims due to its ability to oxygenate, or functionalize, a wide variety of aliphatic and aromatic molecules. This characteristic has been utilized for co-metabolic production processes where methanotrophs are fed both methane and a more complex molecule to be transformed by the sMMO. Numerous examples are reported of processes requiring both methane and, typically, a petroleum-derived feedstock such as toluene, naphthalene, or decane, where sMMO plays a role. However, the art is silent with respect to using methanotrophs for net synthesis of chemicals from methane as opposed to these co-metabolic transformations. For net synthesis, only inexpensive methane is required along with the ability to genetically engineer the strain to produce the desired chemical.
Methanotrophic cells can further build the oxidation products of methane (i.e. methanol and formaldehyde) into more complex molecules such as protein, carbohydrate and lipids. For example, under certain conditions methanotrophs are known to produce exopolysaccharides (Ivanova et al., Mikrobiologiya (1988), 57(4), 600-5; Kilbane, John J., II Gas, Oil, Coal, Environ. Biotechnol. 3, [Pap. IGT""s Int. Symp.], 3rd (1991), Meeting Date 1990, 207-26. Editor(s): Akin, Cavit; Smith, Jared. Publisher: IGT, Chicago, Ill.). Similarly, methanotrophs are known to accumulate both isoprenoid compounds and carotenoid pigments of various carbon lengths (Urakami et al., J. Gen. Appl. Microbiol. (1986), 32(4), 317-41). Although these compounds have been identified in methanotrophs, they have not been microbial platforms of choice for production as these organisms have very poorly developed genetic systems, thereby limiting metabolic engineering for chemicals.
A necessary prerequisite to metabolic engineering of methanotrophs is a full understanding, and optimization, of the carbon metabolism for maximum growth and/or product yield. Obligate methanotrophs are typically thought to channel carbon from methane to useful products and energy via the Entner-Douderoff Pathway which utilizes the keto-deoxy phosphogluconate aldolase enzyme (Dijkhuizen, L., P. R. Levering, G. E. DeVries 1992. In: Methane and Methanol Utilizers (Biotechnology Handbooks 5) J. Colin Murrell and Howard Dalton eds. 1992 Pleanum Press NY pp 149-181). This pathway is not energy-yielding as is the case for the Embden-Meyerhof pathway. Thus, utilization of the Entner-Douderoff pathway results in lower cellular production yields and a greater proportion of the carbon produced as carbon dioxide compared to organisms that use the Embden-Meyerhof pathway. Therefore, a more energy efficient carbon processing pathway would greatly enhance the commercial viability of a methanotrophic platform for the generation of materials.
As noted above, methanotrophic bacteria possess the potential to be commercially effective production platforms for materials such as single cell protein, exopolysaccharides, and long chain carbon molecules such as isoprenoids and carotenoid pigments. The usefulness of methanotrophs for production of a larger range of chemicals is constrained however, by several limitations including, relatively slow growth rates of methanotrophs, limited ability to tolerate methanol as an alternative substrate to methane, difficulty in genetic engineering, poor understanding of the roles of multiple carbon assimilation pathways present in methanotrophs, and potentially high costs due to the oxygen demand of fully saturated substrates such as methane. The problem to be solved therefore is to develop a fast-growing, high yielding methanotroph capable of receiving foreign genes via standard genetic procedures. Full and rapid resolution of central carbon pathways is essential for enabling pathway engineering and carbon flux management for new products.
Applicants have solved the stated problem by providing a methanotrophic bacterial strain capable of efficiently using either methanol or methane as a carbon substrate. The strain is also metabolically versatile in that it contains multiple pathways for the incorporation of carbon from formaldehyde into 3-C units. The discovery of a phosphofructokinase and fructose 1,6 bisphosphate aldolase in this strain suggests that it can utilize the more energetically favorable Embden-Meyerhof pathway in addition to the Entner-Douderoff pathways. The present strain is shown to be useful for the production of a variety of materials beyond single cell protein to include carbohydrates, pigments, terpenoid compounds and aromatic compounds. The formation of large amounts of carbohydrates from methane or methanol can be carried out by this strain. This is surprising and also enables this strain to be used for the production of typical carbohydrate or sugar fermentation end-products such as alcohols, acids and ketones. The present strain was also shown to be capable of genetic exchange with donor species such as Escherichia coli via a standard genetic procedure known as bacterial conjugation. In this way, the strain can be engineered for net synthesis from methane to produce new classes of products other than those naturally produced.
The present invention provides a methanotrophic bacterial strain capable of growth on a C1 carbon substrate. The instant bacterial strain may be further characterized by the ability to grow rapidly and efficiently on either methanol or methane as a sole carbon source. This efficiency is due to the presence of a pyrophosphate linked phosphofructokinase enzyme within an operative Embden-Meyerhof pathway. This is a novel observation for methanotrophic bacteria. Functionally, the utilization of the Emben-Meyerhof pathway and pyrophosphate, instead of the Entner-Douderoff pathway reaction results in highly favorable cellular energetics which is manifested in higher yields, carbon conversion efficiency and growth rate.
The present strain also contains an enzyme system capable of reducing nitrate or nitrite with formation of gaseous nitrogen oxides. This capability is useful for reducing oxygen demand as well as for removing nitrates and nitrites in methane-containing environments such as landfills, wastewater treatment systems or anywhere that methane, oxygen and nitrates are present.
The ability to form large amounts of carbohydrates in the form of starch, polyglucose and/or extracellular polysaccharide is also useful for the production of carbohydrate-based products. Additionally Methylomonas 16a is only capable of growth on methane or methanol and is incapable of proliferating in the human body and thus is completely harmless and non-pathogenic. These characteristics make the strain ideally useful for the production of a wide range of products including animal feeds comprising variable carbohydrate/protein ratios.
The strain is shown to be capable of genetic exchange and expression of foreign genes. Additionally the present strain may be identified by the characteristic 16sRNA sequence as set forth in SEQ ID NO:81.
Additionally the present invention provides methods for the production of single cell protein, carbohydrates, and carotenoid pigments, or higher value mixtures of protein, pigments and carbohydrates. Additionally the strain may be used as a denitrifying agent for the conversion of nitrate or nitrite to nitrous oxide with methane or methanol as carbon source.
Accordingly the invention provides a high growth methanotrophic bacterial strain which:
(a) grows on a C1 carbon substrate selected from the group consisting of methane and methanol; and
(b) comprises a functional Embden-Meyerhof carbon pathway, said pathway comprising a gene encoding a pyrophosphate dependent phosphofructokinase enzyme, the gene selected from the group consisting of:
(a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:6;
(b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS;
(c) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 437 amino acids that has at least 63% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:6; and
(d) an isolated nucleic acid molecule that is complementary to (a), (b) or (c).
Optionally the present strain may comprise at least one gene encoding a fructose bisphosphate aldolase enzyme as part of the functional Embden-Meyerhof carbon pathway. Additionally, the present strain may optionally contain a functional Entner-Douderoff carbon pathway, where the Entner-Douderoff carbon pathway comprises at least one gene encoding a keto-deoxy phosphogluconate aldolase.
In one embodiment the present strain may optionally contain other carbon flux genes encoding polypeptides selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
In another embodiment the present strain may possess a denitrification pathway where the pathway may optionally comprise genes encoding polypeptides having the amino acid sequences selected from the group consisting of SEQ ID NO:40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.
In another embodiment the present strain may contain a set of exopolysaccharide synthesizing enzymes where the exopolysaccharide synthesizing enzymes may have the amino acid sequences selected from the group consisting of SEQ ID NO:22, 24, 26, 28, 30, 32, 34, 36, and 38.
In a more specific embodiment the present strain may comprise genes encoding isoprenoid synthesizing enzymes where the enzymes are selected from the group consisting of SEQ ID NO:62, 64, 66, 68, 70, 72, 74, 86, and 78.
In a preferred embodiment the invention provides a method for the production of single cell protein comprising:
a) contacting the present high growth methanotrophic bacterial strain with a C1 carbon substrate, selected from the group consisting of methane and methanol, in a suitable medium for a time sufficient to permit the expression and accumulation of single cell protein; and
b) optionally recovering the single cell protein.
It is an additional object of the invention to provide a method for the biotransformation of a nitrogen containing compound selected from the group consisting of ammonia, nitrate, nitrite, and dinitrogen comprising, contacting the present high growth methanotrophic bacterial strain with a C1 carbon substrate selected from the group consisting of methane or methanol, in the presence of the nitrogen containing compound, in a suitable medium for a time sufficient to permit the biotransformation of the nitrogen containing compound.
Similarly it is an object of the present invention to provide a method for the production of a feed product comprising protein, carbohydrates and pigment comprising the steps of:
a) contacting the high growth methanotrophic bacterial strain of the present invention with a C1 carbon substrate in a suitable medium for a time sufficient to permit the expression and accumulation of the feed product; and
b) optionally recovering the feed product.
Optionally the relative compositions of protein, carbohydrate and pigment are altered through the up-regulation or down-regulation of any one of the genes encoding the proteins selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69.
In a preferred embodiment the invention provides a method of identifying a high growth methanotrophic bacterial strain comprising:
(a) growing a sample suspected of containing a high growth methanotrophic bacterial strain on a suitable growth medium in the presence of methane as a sole carbon source;
(b) identifying colonies that grow under the conditions of step (a);
(c) analyzing the colonies identified in step (b) for the presence of pyrophosphate dependent phosphofructokinase activity.